1. Field of the Invention
Aspects of the present invention relate generally to the field of microscopy and, more specifically, to methods and apparatuses for reducing background fluorescence in fluorescence microscopy.
2. Description of Related Art
The most common form of fluorescence microscope is an epifluorescence microscope. In this context, “epifluorescence” generally implies that the incidence illumination comes from the same direction or side of the illuminated sample from which fluorescence is detected. Of particular interest is the epifluorescence microscope configuration in which the excitation and the collection or emission beams both pass through the backside of the microscope coverslip, slide, or other optical substrate, upon which the sample being viewed or imaged is located.
A common problem when working with optical substrates, such as glass microscope slides or plastic microtiter plate, is that the backside of the optical substrate becomes contaminated with fluorescent material. Fluorescent contaminants located on the backside of an optical substrate are out of focus relative to the fluorescence of interest and tend to cover or interfere with the fluorescence of interest over a large area. Cleaning fluorescent contaminants from the backside of the optical substrate is very difficult, requiring extra labor and handling of the substrate; increased potential of fouling the sample of interest; slower processing times; and increased costs. In some cases, the fluorescent contaminants are embedded in the surface of the optical substrate and may be impossible to remove using practical, safe solvents and cleaning agents.
In addition to the problem of illuminating out-of-focus contaminants, a second problem lies with background fluorescence arising from the auto-fluorescence of the optical substrate material itself. Even transparent materials such as glass or plastic can produce a detectable amount of fluorescence when the material is thick enough. The fundamental problem is that fluorescence intensity is proportional to the amount of material. Because a typical microscope slide is 1 mm thick, the total thickness of the slide is about two to three orders of magnitude thicker than the target of interest (about 1-10 um thick). From a very simple perspective, the fluorescent probe used to label the target must be at least 300× more fluorescent than the glass in order to achieve a 3:1 signal-to-background ratio. In other words, a large amount of background fluorescence from the substrate will degrade the performance of the instrument by reducing the signal-to-background ratio, which is a critical performance characteristic.
Various microscopy systems and techniques have been employed to reduce or avoid the problems associated with background fluorescence on optical substrates including: laser scanning systems; wide-field imaging systems; evanescent illumination systems; and wide-field systems with oblique illumination. Laser scanning systems have been able to minimize backside problems by illuminating only a single point at a time. The disadvantages of laser scanning systems when attempting to build highly reliable and highly repeatable diagnostic applications are well known. Laser scanning systems are not practical for all types of instruments and have limited ability to perform diagnostic assays.
Wide-field imaging systems that use epifluorescent illumination are the most vulnerable to backside contamination, because the path of the illumination beam exactly matches the path of the image collection optics. The illumination beam passes directly through the backside of the substrate, in the opposite direction of the image collection rays. Similarly, the illumination beam strikes the entire thickness of the substrate on its way to the target.
Evanescent wave illumination is another alternative illumination method. The illumination is directed to the surface of the glass from outside the field-of-view. When illuminated at the so-called “critical angle”, the illumination beam will travel through an evanescent wave along the surface of the glass. The strength of this technique is that only objects along the surface of the glass are illuminated. The weaknesses of this technique include: dim, inefficient illumination; many fluorescent probes are not illuminated because the illuminated region is thin; difficult to align and maintain, especially in a commercial instrument; propagation of the illumination is dependent on surface cleanliness; and illumination shadowing will affect fluorescent signal strength. Evanescent illumination has only been practiced in situations involving laboratory research and is not a promising technique for general purpose fluorescence assays.
Wide-field systems with oblique illumination have some ability to avoid illumination of the backside contamination. The illumination beam comes from outside the cone angle of the objective lens. Many of the illumination rays reach the target without passing through the region of the substrate that is viewed by the objective. When using a simple, planar substrate like a microscope slide, however, it is nearly impossible to avoid significant illumination of the substrate. Backside illumination and bulk material illumination can still limit instrument performance. For example, the wide-field, oblique illumination scanner ArraryWoRx, designed and manufactured by Applied Precision, the assignee of the present invention, unavoidably illuminates one or more optical substrate surfaces that are not in the object plane of the scanner. The backside of the target is partially illuminated, causing undesired background fluorescence. To avoid such background fluorescence it is necessary to clean the backside of the scan target or optical substrate. Because the backside surface is part of the object to be scanned, the cleanliness is difficult to control. The quality of the scanned image is overly dependent on the fabricator of the optical substrate and also the instrument operator.
What is needed in the field is a means to eliminate or avoid illumination of out-of-focus surfaces and fluorescent contaminants located on optical substrates.